Motor neuron degeneration promotes neural progenitor cell proliferation, migration, and neurogenesis in the spinal cords of amyotrophic lateral sclerosis mice

The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter-controlled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by bromodeoxyurindine incorporation and LacZ staining was restricted to the ependymal zone surrounding the central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALS-like mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS.     

Temporal response of neural progenitor cells to disease onset and progression in amyotrophic lateral sclerosis-like transgenic mice

Regenerative medicine through neural stem cells (NSCs) or neural progenitor cells (NPCs) has been proposed as an alterative avenue for restoring neurological dysfunction in amyotrophic lateral sclerosis (ALS). It is critical to understand the organization and distribution of endogenous adult NPCs in response to motor neuron degeneration before regenerative medicine can be applied for ALS therapy. For this reason, we analyzed the temporal response of NPCs to motor neuron degeneration in the spinal cord and brain using nestin enhancer-driven LacZ reporter transgenic mice (pNes-Tg mice, control) and bi-transgenic mice containing both the nestin enhancer-driven LacZ reporter gene and mutant G93A-SOD1 gene (Bi-Tg mice). We observed an increase of NPCs in the dorsal horns of the spinal cord at the disease onset and progression stages in the Bi-Tg mice compared with that of age-matched pNes-Tg control mice. In contrast, an increase of NPCs in the ventral horns was detected at the disease progression stage. On the other hand, an increase of NPCs in the motor cortex at the disease-onset stage, but not at the disease progression stage, was detected. Furthermore, a decrease of NPCs in the lateral ventricle at the disease progression stage was observed, whereas no difference in the number of NPCs in the hippocampus was detected at the disease onset and progression stages. Some of the NPCs differentiate into neuron-like cells in response to motor neuron degeneration. The organization and distribution of endogenous adult NPCs in the ALS-like transgenic mice at the disease onset and progression stages provide fundamental bases for consideration of regenerative therapy of ALS by increasing de novo neurogenesis.     

AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the brain stem and the spinal cords. One of the causes for the familial ALS has been attributed to the mutations in copper-zinc superoxide dismutase (SOD1). Although the toxic function of the mutant enzyme has not been fully understood, the final cell death pathway has been suggested as caspase-dependent. In the present study, we present evidence that the activation of apoptosis inducing factor (AIF) may play a role to induce motor neuron death during ALS pathogenesis. In the spinal cord of SOD1 G93A transgenic mice, expression of AIF was detected in the motor neurons and astrocytes. The level of AIF expression increased as the disease progressed. In the symptomatic SOD1 G93A transgenic mice, AIF released from the mitochondria and translocated into the nucleus in the motor neurons as evidenced by confocal microscopy and biochemical analysis. These results suggest that AIF may play a role to induce motor neuron death in a mouse model of ALS.     

Human superoxide dismutase 1 overexpression in motor neurons of Caenorhabditis elegans causes axon guidance defect and neurodegeneration

Strong evidence indicates that mutant Cu, Zn-superoxide dismutase 1 (SOD1) exerts toxic effect on motor neurons in amyotrophic lateral sclerosis (ALS). However, the nature of mutant SOD1-mediated motor neuron degeneration is poorly understood. To provide new insight into the mechanism by which mutant SOD1 induces motor neuron injury, we developed novel Caenorhabditis elegans models of ALS. Expression of human wild type or G93A SOD1 specifically in motor neurons of C. elegans caused progressive locomotion defect and paralytic phenotype, which recapitulate some characteristic features of ALS including age-dependent motor dysfunction and degeneration of motor neurons associated with SOD1 aggregation. In addition, the motor neuron loss is independent of cell death protein 3 (CED-3)/cell death protein 4 (CED-4) caspase pathway. We also found that before motor neurons began to die in adulthood, axon guidance defect of motor neuron appeared during the development stages. When green fluorescent protein (GFP)-tagged proteins related to axon guidance were examined in motor neurons, a significantly decreased density and number of GFP-tagged puncta were observed in the transgenic worms. Our models mimic axon developmental defect and the adult-onset degeneration of motor neurons in ALS. Using this model, we uncovered the cell-autonomous damage caused by human SOD1 to motor neurons in vivo, and provided a new insight into the developmental defect mechanism that may contribute to motor neuron degeneration in ALS.     

Lack of caspase-dependent apoptosis in spinal motor neurons of the wobbler mouse

The mechanism of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is still unclear and the post-mortem analysis of samples from ALS patients does not permit a clarification of the early events of cell death occurring in ALS. Animal models of motor neuron degeneration represent a reliable tool to investigate the type of cell death. Attention was focused on the possible role of apoptosis in a spontaneous model of cervical spinal cord motor neuron degeneration, the wobbler mouse. Firstly, the rate of motor neuron loss occurring in the cervical spinal cord region of wobbler mice during different phases of symptoms progression was quantified by CholineAcetyltransferase (ChAT) immunohistochemistry. This was followed by a series of immunohistological studies to ascertain whether apoptosis was actually involved. ChAT immunostaining confirmed the severe loss of cholinergic neurons. Since the rate of motor neuron death is maximal in the first phase of the disease (from the 3rd to the 5th postnatal week), apoptotic markers were evaluated in 4-week-old wobbler mice. This study, carried out by examining a large number of cervical spinal cord sections from 20 affected animals and 20 healthy littermates, did not show either caspase activation or DNA fragmentation. These results strongly suggest that motor neuron death occurring in the wobbler mouse is not related to a caspase-dependent apoptotic mechanism.     

C3H1型锌指结合蛋白36介导的星形胶质细胞活化在肌萎缩侧索硬化症运动神经元退变中的作用

目的 探讨C3H1型锌指结合蛋白36（ZFP36L1）介导的星形胶质细胞活化在肌萎缩侧索硬化症（ALS）运动神经元退变中的作用。 方法 以铜锌超氧化物歧化酶1（SOD1)-G93A转基因小鼠作为动物模型,同窝野生型小鼠作为对照（突变型及野生型小鼠各时间点分别取13只小鼠）;Real-time PCR、Western blotting检测小鼠发病早期、中期及晚期脊髓内ZFP36L1 mRNA及蛋白变化,免疫荧光染色检测ZFP36L1在脊髓内的表达及分布;出生后 1～2 d新生小鼠15只,建立SOD1-G93A突变型原代星形胶质细胞模型,Real-time PCR、Western blotting检测星形胶质细胞内ZFP36L1 mRNA及蛋白水平的变化,si-ZFP36L1转染SOD1-G93A突变型原代星形胶质细胞,Western blotting检测转染效率,Western blotting及ELISA检测转染后星形胶质细胞分泌炎性因子肿瘤坏死因子α（TNF-α）、白细胞介素18（IL-18）变化;沉默SOD1-G93A突变型原代星形胶质细胞内ZFP36L1后,与SOD1-G93A突变型NSC34细胞共培养,通过5’-乙炔基-2’-脱氧尿苷（EdU）实验和观察增殖细胞核抗原（PCNA）的水平明确ZFP36L1对NSC34细胞增殖的影响,通过TUNEL实验及观察剪切Caspase-3（cleaved-Caspase-3）的水平明确ZFP36L1对NSC34细胞凋亡的影响,以转染空白小干扰RNA（siRNA）作为对照组。 结果 与野生型小鼠相比,ZFP36L1在SOD1-G93A转基因小鼠脊髓组织内mRNA及蛋白水平均下调,在野生型小鼠脊髓组织内,ZFP36L1主要与β-微管蛋白Ⅲ（β-tubulin Ⅲ）阳性的神经元共表达,而SOD1-G93A突变型小鼠的脊髓组织内,ZFP36L1在神经元内表达减弱,与胶质纤维酸性蛋白（GFAP）标记的星形胶质细胞共表达明显增加;SOD1-G93A突变型原代星形胶质细胞内ZFP36L1表达增加,si-ZFP36L1能明显降低SOD1-G93A突变型原代星形胶质细胞内ZFP36L1水平;沉默ZFP36L1后星形胶质细胞分泌炎性因子 TNF-α、 IL-18明显降低。此外,沉默ZFP36L1后,SOD1-G93A突变型原代星形胶质细胞能显著增强NSC34细胞增殖活性,抑制NSC34细胞凋亡。 结论 在ALS发病过程中,星形胶质细胞被激活,ZFP36L1通过星形胶质细胞分泌的炎性因子促进了ALS运动神经元的退变。     

Mechanisms for neuronal degeneration in amyotrophic lateral sclerosis and in models of motor neuron death (Review)

Amyotrophic lateral sclerosis (ALS), also referred to as motor neurone disease, is a fatal neurological disease that is characterized clinically by progressive muscle weakness, muscle atrophy, and eventual paralysis. The neuropathology of ALS is primary degeneration of upper (motor cortical) and lower (brainstem and spinal) motor neurons. The amyotrophy refers to the neurogenic atrophy of affected muscle groups, and the lateral sclerosis refers to the hardening of the lateral white matter funiculus in spinal cord (corresponding to degeneration of the corticospinal tract) found at autopsy. Because the mechanisms for the motor neuron degeneration in ALS are not understood, this disease has no precisely known causes and no effective treatments. Very recent studies have identified that the degeneration of motor neurons in ALS is a form of apoptotic cell death that may occur by an abnormal programmed cell death (PCD) mechanism. In order to treat ALS effectively, we need to understand the mechanisms for motor neuron apoptosis more completely. Future studies need to further identify the signals for PCD activation in neurons as they relate to the pathogenesis of ALS and to clarify the molecular pathways leading to motor neuron apoptosis in animal and cell culture model systems. These studies should lead to a better understanding of motor neuron death and to the design of new therapeutic experiments critical for the future treatment of ALS.     

Topiramate protects against motor neuron degeneration in organotypic spinal cord cultures but not in G93A SOD1 transgenic mice

Topiramate is a novel anti-convulsant, structurally distinct from other known anti-convulsants. A number of independent studies suggest that topiramate has anti-excitotoxic properties. It has been found to diminish release of glutamate from neurons and block (-amino-3-hydoxy-5-methylisoxazole-4-proprionic acid glutamate receptor evoked currents. Since activation of non-N-methyl-D-aspartate glutamate receptors is thought to play a role in the selective loss of motor neurons in amyotrophic lateral sclerosis (ALS), we determined whether topiramate could protect against chronic glutamate-mediated motor neuron degeneration. An organotypic spinal cord culture system was used in which glutamate transport is inhibited by pharmacological blockade. After 3 weeks of treatment, topiramate was found to significantly prevent motor neuron degeneration in this culture model. However, the drug did not increase survival in G93A SOD1 transgenic mice, an animal model of ALS. These studies suggest that topiramate could be useful as a neuroprotectant, but were not effective in more complex motor injury paradigms such as the mouse model of ALS.     

Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice

The glial glutamate transporter EAAT2 is primarily responsible for clearance of glutamate from the synaptic cleft and loss of EAAT2 has been previously reported in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. The loss of functional EAAT2 could lead to the accumulation of extracellular glutamate, resulting in cell death known as excitotoxicity. However, it is still unknown whether it is a primary cause in the cascade leading to neuron degeneration or a secondary event to cell death. The goals of this study were to generate transgenic mice overexpressing EAAT2 and then to cross these mice with the ALS-associated mutant SOD1(G93A) mice to investigate whether supplementation of the loss of EAAT2 would delay or rescue the disease progression. We show that the amount of EAAT2 protein and the associated Na+-dependent glutamate uptake was increased about 2-fold in our EAAT2 transgenic mice. The transgenic EAAT2 protein was properly localized to the cell surface on the plasma membrane. Increased EAAT2 expression protects neurons from L-glutamate induced cytotoxicity and cell death in vitro. Furthermore, our EAAT2/G93A double transgenic mice showed a statistically significant (14 days) delay in grip strength decline but not in the onset of paralysis, body weight decline or life span when compared with G93A littermates. Moreover, a delay in the loss of motor neurons and their axonal morphologies as well as other events including caspase-3 activation and SOD1 aggregation were also observed. These results suggest that the loss of EAAT2 may contribute to, but does not cause, motor neuron degeneration in ALS.     

Protective effects of resveratrol through the up-regulation of SIRT1 expression in the mutant hSOD1-G93A-bearing motor neuron-like cell culture model of amyotrophic lateral sclerosis

Resveratrol has recently been widely reported to be an age-delaying and neuroprotective compound, and it appears to produce these benefits by activating silent mating type information regulation 2 homolog 1 (SIRT1). However, the role that SIRT1 activation plays in the pathogenesis of amyotrophic lateral sclerosis (ALS) remains unclear. In the present study, SIRT1 expression was found to be much lower in the mutant hSOD1G93A-bearing VSC4.1 cells compared to hSOD1wt cells when both were cultured in low-serum medium, indicating the involvement of SIRT1 activation defects in the pathogenesis of ALS under energetic stress. Further investigation revealed that a 24-h treatment with 0.5-20 μM resveratrol had a dose-dependent protective effect on this ALS cell model, and the effects of resveratrol on increasing cell viability, preventing cell apoptosis and elevating cellular ATP levels through promoting mitochondria biogenesis were blocked by SIRT1 inhibition. This further demonstrated a role for SIRT1 activation in the protection of neuronal cells from degeneration. These findings suggest that resveratrol can protect the ALS cell model from mutant SOD1-mediated toxicity through up-regulating the expression of SIRT1, which represents a potential therapeutic target for preventing the motor neuron degeneration in ALS patients.     

Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease

Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction also occurs in human ALS and transgenic rodents expressing mutant human SOD1 protein (SOD1(G93A)). Here we investigated focal enrichment of normal astrocytes using transplantation of lineage-restricted astrocyte precursors, called glial-restricted precursors (GRPs). We transplanted GRPs around cervical spinal cord respiratory motor neuron pools, the principal cells whose dysfunction precipitates death in ALS. GRPs survived in diseased tissue, differentiated efficiently into astrocytes and reduced microgliosis in the cervical spinal cords of SOD1(G93A) rats. GRPs also extended survival and disease duration, attenuated motor neuron loss and slowed declines in forelimb motor and respiratory physiological functions. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter GLT1. These findings indicate the feasibility and efficacy of transplantation-based astrocyte replacement and show that targeted multisegmental cell delivery to the cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.     

Altered distribution and levels of cathepsinD and cystatins in amyotrophic lateral sclerosis transgenic mice: possible roles in motor neuron survival

In amyotrophic lateral sclerosis (ALS) there is a selective degeneration of motor neurons leading to muscle paralysis and death. The mechanism underlying cell demise in ALS is not fully understood, but involves the activation of different proteolytic enzymes, including the caspase family of cysteine proteases. We have here studied whether other proteases, such as the cathepsins, residing in lysosomes, and the cathepsin inhibitors, cystatinB and -C are changed in ALS. The expression and protein levels of the cathepsinB, -L and -D all increased in the spinal cord in ALS mice, carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. At the cellular level, cathepsinB and -L were present in ventral motor neurons in controls, but in the ALS mice cathepsinB was also expressed by glial fibrillary acidic protein (GFAP) positive astrocytes. The distribution of the aspartic protease, cathepsinD also changed in ALS with a loss of the lysosomal staining in motor neurons. Inhibition of caspases by means of X-chromosome-linked inhibitor of apoptosis protein (XIAP) overexpression did not inhibit cleavage of cathepsinD in ALS mice, suggesting a caspase-independent pathway. Expression of cystatinB and -C increased slightly in the ALS spinal cords. Immunostaining showed that in ALS, cystatinC was present in motor neurons and in GFAP positive astrocytes. CystatinB that is a neuroprotective factor decreased in motor neurons in ALS but was expressed by activated microglial cells. The observed changes in the levels and distributions of cathepsinD and cystatinB and-C indicate a role of these proteins in the degeneration of motor neurons in ALS.     

Deficiency in the ALS2 gene does not affect the motor neuron degeneration in SOD1(G93A) transgenic mice

Dysfunction of the ALS2 gene has been linked to one form of juvenile onset autosomal recessive amyotrophic lateral sclerosis (ALS). Previous in vitro studies suggest that over-expression of ALS2 protects cells from mutant Cu/Zn superoxide dismutase (SOD1)-induced cytotoxicity. To test whether ALS2 plays a protective role against mutant SOD1-mediated motor neuron degeneration in vivo, we examined the progression of motor neuron disease in SOD1(G93A) mice on an ALS2 null background. Our data suggest that deficiency in the ALS2 gene does not affect the pathogenesis of SOD1(G93A) mice.     

ALS-linked mutant SOD1 induces ER stress- and ASK1-dependent motor neuron death by targeting Derlin-1

Mutation in Cu/Zn-superoxide dismutase (SOD1) is a cause of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 protein (SOD1(mut)) induces motor neuron death, although the molecular mechanism of SOD1(mut)-induced cell death remains controversial. Here we show that SOD1(mut) specifically interacted with Derlin-1, a component of endoplasmic reticulum (ER)-associated degradation (ERAD) machinery and triggered ER stress through dysfunction of ERAD. SOD1(mut)-induced ER stress activated the apoptosis signal-regulating kinase 1 (ASK1)-dependent cell death pathway. Perturbation of binding between SOD1(mut) and Derlin-1 by Derlin-1-derived oligopeptide suppressed SOD1(mut)-induced ER stress, ASK1 activation, and motor neuron death. Moreover, deletion of ASK1 mitigated the motor neuron loss and extended the life span of SOD1(mut) transgenic mice. These findings demonstrate that ER stress-induced ASK1 activation, which is triggered by the specific interaction of Derlin-1 with SOD1(mut), is crucial for disease progression of familial ALS.     

Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons.

We have developed a series of mouse-mouse neural hybrid cell lines by fusing the aminopterin-sensitive neuroblastoma N18TG2 with motor neuron-enriched embryonic day 12-14 spinal cord cells. Of 30 neuroblastoma-spinal cord (NSC) hybrids displaying a multipolar neuron-like phenotype, 10 express choline acetyltransferase, and 4 induce twitching in cocultured mouse myotubules. NSC-19, NSC-34, and their subclones express additional properties expected of motor neurons, including generation of action potentials, expression of neurofilament triplet proteins, and acetylcholine synthesis, storage, and release. In addition, NSC-34 cells induce acetylcholine receptor clusters on cocultured myotubes, and undergo a vimentin-neurofilament switch with maturation in culture, similar to that occurring in neuronal development. NSC cell lines appear to model selected aspects of motor neuron development in an immortalized clonal system.     

Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model

Here we report an in vitro model system for studying the molecular and cellular mechanisms that underlie the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Embryonic stem cells (ESCs) derived from mice carrying normal or mutant transgenic alleles of the human SOD1 gene were used to generate motor neurons by in vitro differentiation. These motor neurons could be maintained in long-term coculture either with additional cells that arose during differentiation or with primary glial cells. Motor neurons carrying either the nonpathological human SOD1 transgene or the mutant SOD1(G93A) allele showed neurodegenerative properties when cocultured with SOD1(G93A) glial cells. Thus, our studies demonstrate that glial cells carrying a human SOD1(G93A) mutation have a direct, non-cell autonomous effect on motor neuron survival. More generally, our results show that ESC-based models of disease provide a powerful tool for studying the mechanisms of neural degeneration. These phenotypes displayed in culture could provide cell-based assays for the identification of new ALS drugs.     

Lack of TDP-43 abnormalities in mutant SOD1 transgenic mice shows disparity with ALS

Mislocalization of the TAR-DNA binding protein (TDP-43) from the nucleus to the cytoplasm of diseased motor neurons and association with intraneuronal ubiquitinated inclusions has recently been reported in amyotrophic lateral sclerosis (ALS). Here, we have investigated TDP-43 immunoreactivity in three lines of mutant SOD1 transgenic mice, G93A, G37R and G85R and compared with labeling in one sporadic ALS case and two familial ALS cases carrying mutations in SOD1, A4T and I113T. Our findings show that there is no mislocalization of TDP-43 to the cytoplasm in motor neurons of mutant SOD1 transgenic mice, nor association of TDP-43 with ubiquitinated inclusions. In contrast, mislocalization of TDP-43 to the cytoplasm and association with ubiquitinated inclusions was found in the ALS cases, including those carrying mutations in SOD1. Interestingly, there was no association of TDP-43 with ubiquitinated hyaline conglomerate inclusions, pathology closely associated with ALS cases carrying mutations in SOD1. Our findings indicate that the process of motor neuron degeneration in mutant SOD1 transgenic mice is unlikely to involve the abnormalities of TDP-43 described in the human disease.     

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1^G93A mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1^G93A mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1^G93A mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.